The present invention relates generally to lymphokines, and particularly to pharmaceutical compositions comprising selected truncated nonglycosylated analog interleukin-3 (IL-3) proteins which exhibit beneficial clinical and toxicological properties when compared to huIL-3 proteins produced in E. coli.
The differentiation and proliferation of hematopoietic cells is regulated by secreted glycoproteins collectively known as colony stimulating factors (CSFs). In murine and human systems, these proteins include granulocyte-macrophage colony stimulating factor (GM-CSF), which promotes granulocyte and macrophage production from normal bone marrow, and which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. Other CSFs include macrophage CSF (M-CSF or CSF-1), which induces the selective proliferation of macrophages, and granulocyte CSF (G-CSF) which induces development of granulocyte progenitors from bone marrow precursors. An additional CSF, isolated first in murine systems and more recently from human cell sources, has been designated IL-3 or multi-CSF.
Murine IL-3 was originally identified by Ihle et al., J. Immunol. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20.alpha.-hydroxysteriod dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells. cDNA clones corresponding to murine IL-3 were first isolated by Fung et al., Nature 307:233 (1984) and Yokota et al., Proc. Natl. Acad. Sci. USA 81:1070 (1984). Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by Yang et al., Cell 47:3 (1986). The human sequence reported by Yang et al. included a serine residue at position 8 of the mature protein sequence. Following this finding, three groups reported isolation of Pro.sup.8 huIL-3 cDNAs, including Dorssers et al., Gene 55:115 (1987); Otsuka et al., J. Immunol. 140:2288 (1988); and Gillis et al., Behring Inst. Mitt. 83:1 (1988).
A survey of individuals to determine the frequency of the Pro.sup.8 allele was reported by Gillis et al., supra. In this work, the polymerase chain reaction was employed to amplify the DNA sequences flanking the position 8 locus. Radiolabeled oligonucleotide probes complementary to the Ser.sup.8 and Pro.sup.8 forms were then used to probe amplified DNA isolated from thirteen genetically unrelated individuals. Amplified DNA was immobilized on nitrocellulose and analyzed by hybridization under conditions of increasing stringency. The results indicated that of the 13 persons examined, each was positive for DNA encoding the Pro.sup.8 version of huIL-3. Three were also positive for Ser.sup.8 huIL-3 at this locus (i.e., were heterozygotes), indicating a moderate level of polymorphism of the human IL-3 gene in this test population.
Preclinical and clinical studies of human IL-3 proteins produced by various manufacturers has revealed surprising differences in clinical utility and toxicity. Development of non-toxic, tolerable forms of human IL-3 for therapy is important because recombinant human IL-3 appears to be the first cytokine capable of stimulating granulopoiesis, erythropoiesis and, most important, thrombopoiesis in vivo.
Severe rIL-3 toxicity was reported by Valent et al. at the First International Symposium: Cytokines in Hemopoiesis, Oncology and AIDS, held in Hanover, Federal Republic of Germany, Jun. 14-17, 1989. A composition of huIL-3 (Ser.sup.8) produced in E. coli was evaluated in vivo by administration to rhesus monkeys (n=10) thrice daily at different dosages (0, 11, 33, and 100 .mu.g/kg/day, s.c.) for 14 days followed by consecutive GM-CSF application (days 14-28, 5 .mu.g/kg thrice daily). All monkeys responded to rhuIL-3 by a 2-3 fold white blood cell (WBC) increase by day 14 due to an increase in eosinophils and basophils. Intracellular histamine (IH) in WBC was found to rise continuously after IL-3 treatment until day 8-10. This excess of IH was accompanied by a rise in plasma histamine values and an urticaria-like exanthem revealing infiltration of mast cells and lymphocytes into the dermis observed in skin biopsy specimens. The clinical side effects of this rhuIL-3 composition were attributed to basophil/mast cell activation and histamine-producing effects of this cytokine.
In contrast, the protein compositions of the present invention did not exhibit any detectable urticaria or stimulate infiltration of mast cells or lymphocytes into the dermis when administered to cynomolgus monkeys or human patients. The results of these toxicological and clinical studies are reported in the Examples.